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Peripheral blood leukocytes of 11 normal cows, 7 cows heterozygous and 2 heifers homozygous for bovine leukocyte adhesion deficiency (BLAD) were analysed by flow cytometry for the intensity of their beta 2 integrin expression (LFA-1(CD11a/CD18), CR3 (CD11b/CD18) and CR4 (CD11c/CD18)). BLAD-homozygotes revealed no or a very weak expression of the beta 2 integrins and had a 10-fold and 4- to 5-fold increase in absolute number of neutrophils and monocytes, respectively, whereas the absolute number of lymphocytes remained normal. The mean fluorescence intensity (MFI) of the beta 2 integrins (CD18) in heterozygous animals was 56 to 90% of this in the normal cows (MFI between 14 and 512). The difference in the expression level was most pronounced for LFA-1 on the small cluster of lymphocytes with the highest MFI for LFA-1. Repeated analysis and phorbol myristate acetate stimulation revealed that the LFA-1 expression on this high-expressing cell population of the peripheral blood allowed a ready identification of BLAD-heterozygotes by flow cytometry.

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The pathological clinical and laboratory findings obtained in 50 calves and young cattle affected with Bovine Leukocyte Adhesion Deficiency are compared with those found in 114 calves and young cattle showing marked neutrophil leukocytosis of other origin (age: < 2 years; leukocyte count: > 30,000 per microl; percentage of lymphocytes: < 55%).

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A PCR-based DNA test for the genetic defect resulting in the deficiency of uridine monophosphate synthase (DUMPS) was developed for embryonic cells to identify the homozygous recessive genotype. This genotype is usually lethal at about day 40 of gestation. Oocytes from a cow, heterozygous for DUMPS, were fertilized in vitro with spermatozoa from a DUMPS-carrier bull and cultured in vitro until the morula or blastocyst stage. Embryos were matured in vitro, fertilized and cultured according to a method established in our laboratory. Heminested PCR was performed and the genotype of twelve embryos were unambiguously determined by the Aval digestion of the PCR products. Among these embryos two individuals homozygous for the defective DUMPS allele (R405-->STOP) were detected. From the remaining ten embryos, four were homozygous and six were heterozygous for the normal allele. The observed distribution is close to the expected ratio of 1:2:1. These results further support the monogenic recessive inheritance of the defect.

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Deficiency of uridine monophosphate synthase (DUMPS) is a monogenic autosomal recessive disorder in cattle, resulting in early embryonic death of homozygous offspring. To identify the mutation responsible for DUMPS, liver RNA from identified, DUMPS heterozygous animals from the Holstein and Red Holstein breeds was reverse transcribed. Amplification of cDNA with sequence-specific primers and subsequent sequencing of the PCR products revealed a mutation (C-->T) with the loss of an AvaI site at codon 405, resulting in a premature stop codon with a truncated C-terminal catalytic subunit of the protein. A direct DNA test based on PCR was developed and subsequently tested on 102 animals. Complete concurrence of deficiency of UMPS and the presence of the described point mutation in heterozygous animals was observed, thus confirming this point mutation as the basic defect in DUMPS cattle.

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A 1869-bp cDNA encoding bovine UMP synthase (UMPS), including the 3'-untranslated and 34 bp of the 5'-untranslated regions, was isolated and sequenced. The deduced amino acid sequence shows a high degree of homology to UMPS sequences reported from other species, namely for regions corresponding to the putative catalytic sites. The sequence information will be used to analyse the molecular basis of the deficiency of UMPS (DUMPS) in cattle.

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Recombinant DNA techniques offer the possibility of diagnosing genetic defects directly by analysing DNA itself. This is especially interesting for detecting carriers of recessive defects. In comparison with phenotypic screening with progeny testing or biochemical tests, DNA screening is independent of the time of gene expression and is not influenced by non-genetic effects. If the mutation causing the defect is known, alterations of DNA sequences can be identified directly as restriction fragment length polymorphisms (RFLPs) or with the use of allele specific oligonucleotides (ASO). DNA amplification with the polymerase chain reaction makes screening tests faster and more accurate. For most defects, the genetic basis is not known. Pedigree analysis with linked polymorphic DNA markers can be used to establish defect diagnosis. Linkage analysis can locate the chromosomal region of the gene responsible for the disease. Identification of the gene and the mutation in the defect allele finally will lead to direct DNA diagnosis. A dense linkage map with highly polymorphic genetic markers covering the entire genome will in future improve understanding of polygenic diseases. Increasing knowledge of the molecular genetics of human defects will promote DNA diagnosis in other species.

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The identification of phenotypically normal carriers of genetic defects is crucial for eliminating recessive defect genes from farm animal populations. Recombinant DNA techniques allow us to identify defect genes on the DNA-basis regardless of the animals age and time of gene expression. Genetic defects also are important as model systems to investigate the regulation of metabolic pathways and the mechanisms of embryonic development.

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